The present invention relates to the provision of a DNA sequence of the major grass pollen allergen Lol p 4. The invention also encompasses fragments, new combinations of partial sequences and point mutants having a hypoallergenic action. The recombinant DNA molecules and the derived polypeptides, fragments, new combinations of partial sequences and variants can be utilised for the therapy of pollen-allergic diseases. The proteins prepared by recombinant methods can be employed for in vitro and in vivo diagnosis of pollen allergies.
Type 1 allergies are of importance worldwide. Up to 20% of the population in industrialised countries suffer from complaints such as allergic rhinitis, conjunctivitis or bronchial asthma. These allergies are caused by allergens present in the air (aeroallergens) which are released by sources of various origin, such as plant pollen, mites, cats or dogs. Up to 40% of these type 1 allergy sufferers in turn exhibit specific IgE reactivity with grass pollen allergens (Freidhoff et al., 1986, J. Allergy Clin. Immunol. 78, 1190-2001).
The substances which trigger type 1 allergy are proteins, glycoproteins or polypeptides. After uptake via the mucous membranes, these allergens react with the IgE molecules bonded to the surface of mast cells in sensitised individuals. If two IgE molecules are crosslinked to one another by an allergen, this results in the release of mediators (for example histamine, prostaglandins) and cytokines by the effector cell and thus in the corresponding clinical symptoms.
A distinction is made between major and minor allergens, depending on the relative frequency with which the individual allergen molecules react with the IgE antibodies of allergy sufferers.
For perennial ryegrass (Lolium perenne), Lol p 1 has been identified as a major allergen (Freidhoff et al., 1986, J. Allergy Clin. 78:1190-1201) and its primary structure has been elucidated (Perez et al., 1990, J. Biol. Chem. 265:16210-16215). A further major allergen is Lol p 2 (Freidhoff et al., 1986, J. Allergy Clin. 78:1190-1201), the primary structure of which was described in 1993 (Ansari et al., 1989, J. Biol. Chem.: 264:11181-11185). A further major allergen of perennial ryegrass is Lol p 5 (Mattiesen and Löwenstein 1991, Clin. Exp. Allergy 21: 297-307). The primary structure of Lol p 5 is also known (Ong et al., 1993, Gene 134:235-240). Perennial ryegrass furthermore contains the major allergens from groups 4 (Fahlbusch et al. 1998, Clin. Exp. Allergy 28: 799-807) and 13 (Petersen et al., 2001, J. Allergy Clin. 1 mm. 107:856-862).
Lol p 4 is a typical basic glycoprotein (Jaggi et al, 1989, Int. Arch. Allergy Appl. Immunol. 89:342-348, Jaggi et al., 1989, J. Allergy Clin. Immunol. 83:845-852) and is comparable with the well-studied Phl p 4, Cyn d 4 and Dac g 4 in terms of cross-reactivity with specific IgE antibodies (Haavik et al., 1985, Int. Arch. Allergy Appl. Immunol. 78:260-268; Su et al., 1991, Clin. Exp. Allergy 21:449-455; Leduc-Brodard et al., 1996, J. Allergy Clin. Immunol. 98:1065-1072; 14-17).
These homologous molecules from the Poaceae form allergen group 4, the molecules of which have high immunological cross-reactivity with one another both with monoclonal murine antibodies and also with human IgE antibodies (Fahlbusch et al., 1993 Clin. Exp. Allergy 23:51-60; Leduc-Brodard et al., 1996, J. Allergy Clin. Immunol. 98:1065-1072; Su et al., 1996, J. Allergy Clin. Immunol. 97:210; Fahlbusch et al., 1998, Clin. Exp. Allergy 28:799-807; Gavrovic-Jankulovic et al., 2000, Invest. Allergol. Clin. Immunol. 10 (6):361-367; Stumvoll et al. 2002, Biol. Chem. 383:1383-1396; Grote et al., 2002, Biol. Chem. 383:1441-1445; Andersson and Lidholm, 2003, Int. Arch. Allergy Immunol. 130:87-107; Mari, 2003, Clin. Exp. Allergy, 33 (1):43-51).
In contrast to the above-mentioned major allergens Lol p 1, Lol p 2, Lol p 5 from Lolium perenne, the primary structure of Lol p 4 has not yet been elucidated.
From the group 4 allergen from Dactylus glomerate, it has hitherto only been possible for peptides to be obtained by enzymatic degradation and sequenced:
DIYNYMEPYVSK (SEQ ID NO 7),
VDPTDYFGNEQ (SEQ ID NO 8),
ARTAWVDSGAQLGELSY (SEQ ID NO 9)
and GVLFNIQYVNYWFAP (SEQ ID NO 10, Leduc-Brodard et al., 1996, J. Allergy Clin. Immunol. 98: 1065-1072).
Peptides have also been obtained from the group 4 allergen of sub-tropical Bermuda grass (Cynodon dactylon) by proteolysis and sequenced:
KTVKPLYIITP (SEQ ID NO 11),
KQVERDFLTSLTKDIPQLYLKS (SEQ ID NO 12),
TVKPLYIITPITAAMI (SEQ ID NO 13),
LRKYGTAADNVIDAKVVDAQGRLL (SEQ ID NO 14),
KWQTVAPALPDPNM (SEQ ID NO 15),
VTWIESVPYIPMGDK (SEQ ID NO 16),
GTVRDLLXRTSNIKAFGKY (SEQ ID NO 17),
TSNIKAFGKYKSDYVLEPIPKKS (SEQ ID NO 18),
YRDLDLGVNQWG (SEQ ID NO 19),
SATPPTHRSGVLFNI (SEQ ID NO 20),
and AAAALPTQVTRDIYAFMTPYVSKNPRQAYVNYRDLD (SEQ ID NO 21, Liaw et al., 2001, Biochem. Biophys. Research Communication 280: 738-743).
For Lolium perenne, peptide fragments having the following sequences have been described for the basic group 4 allergen: FLEPVLGLIFPAGV (SEQ ID NO 22) and GLIEFPAGV (SEQ ID NO 23, Jaggi et al., 1989, Int. Arch. Allergy Appl. Immunol. 89: 342-348).
However, these peptide sequences which have been described for Lolium perenne and other group 4 allergens have hitherto not resulted in the elucidation of the primary structure of the Lol p 4 allergen.
As the first sequence of a group 4 allergen, the still unpublished sequence of Phl p 4 from Phleum pratense has been elucidated by the inventors of the present patent application and described in International Application WO 04/000881.
The object on which the present invention is based therefore consisted in the provision of a DNA sequence of the Lol p 4 gene encoding an allergen having the immunological properties of Lol p 4, and a corresponding recombinant DNA, on the basis of which the allergen can be expressed as protein and made available, as such or in modified form, for pharmacologically significant exploitation. The sequence of Phl p 4 was the starting point for the present, invention.
List of Sequences According to the Invention
                DNA sequence from the Lol p 4 gene (SEQ ID NO 1).        Protein sequence derived from the DNA sequence in accordance with SEQ ID NO 1 (SEQ ID NO 2).        DNA sequence (SEQ ID NO 3), composed of nucleotides 1-200 of Phl p 4 (in accordance with SEQ ID NO 5), 201-1472 of Lol p 4 (in accordance with SEQ ID NO 1) and 1473-1503 of Phl p 4 (in accordance with SEQ ID NO 5).        Protein sequence (SEQ ID NO 4), composed of amino acids 1-67 of Phl p 4 (in accordance with SEQ ID NO 6), 68-490 of Lol p 4 (in accordance with SEQ ID NO 2) and 491-500 of Phl p 4 (in accordance with SEQ ID NO 6) having the properties, in particular immunological properties, of Lol p 4, encoded by the DNA sequence in accordance with SEQ ID NO 3.        DNA sequence of Phl p 4 (SEQ ID NO 5), in accordance with SEQ ID NO 5 from WO 04/000881.        Protein sequence of Phl p 4 (SEQ ID NO 6), in accordance with SEQ ID NO 6 from WO 04/000881.        